I. Field of the Invention
The present invention relates to a method and apparatus for splitting a continuous stream of heterogeneous living cells into numerous separate vessels each containing a single type of homogeneous cells. More particularly, the present invention relates to the use of electronic, high frequency fields combined with gravity pumps to cleanly separate living cells.
II. Description of the Relevant Art
An important facet in the treatment and prevention of disease is the identification, isolation and analysis of cells and cellular material. Once isolated, individual cells can be investigated to better understand cell function and transformation. Clean separation of discrete cell types from a highly mixed heterogeneous population would allow for the removal of one cell type from that population, for example, leukemia cells from blood. Better separation could improve the physician's analysis of blood samples.
While investigation of living cells is undertaken by other means, it is the limitations of cell sorting that has limited cell testing, research and analysis. This is a considerable obstacle, since the subjects under investigation have normally been hard to obtain in adequate quantity and quality. Scientists are still plagued with the inability to cleanly obtain identifiable cell lines in good condition, such as non-toxic, homogeneous, viable forms from a mixture of similar cells for study or removal.
In the mid-1970's a technique called fluorescence-activated cell sorting was developed employing fluorescent dyes (specifically, fluorescein, a dye that glows an intense greenish-blue fluorescence by reflected light, while being reddish-orange by transmitted light). By this method, the dye is mixed with the cells and adheres selectively to different surface features of a particular cell type. The stream of mixed cells, dyed and suspended in fluid, is passed through a rapidly vibrating nozzle that breaks the fluid into small droplets containing individual cells. The stream of individual droplets then passes through a laser beam thus exciting the fluorescein fixed to the cells. By converting the glow to an electrical signal, the fluorescent cells receive a negative charge, the non-fluorescent cells receive a positive charge, and other droplets remain uncharged.
The mixture of charged and uncharged droplets is then passed through an electric field that causes positively charged cells to migrate toward a negative deflection plate, the negatively charged cells to migrate to a positively charged deflection plate, and the uncharged cells to pass between the plates. Collectors are provided to gather groups of separated cells.
While fluorescence-activated cell sorting represented a substantial move in the right direction in providing a method of isolating certain types of cancerous cells and cells of the immune system, this method has several disadvantages. While having relatively low toxicity, fluorescein is still toxic. Its toxicity notwithstanding, the inclusion of a dye in the process adds both another layer of work for the technician and another element to the product. In addition, many different cells respond similarly to the dye and are therefore not separable by this method. Further, all cells are not able to withstand the intense laser light or the high voltages (2,000+ volts) necessary for this type of sorting. Because cell viability is the essential requirement of virtually all cellular investigation and therapy, this sorting method is limited by the low survival rate of the cells.
In the early 1980's, several methods were developed that claimed to avoid the fatal results of the fluorescence-activated cell sorting method. An example is seen in U.S. Pat. No. 4,326,934, issued to Herbert A Pohl for "Continuous Dielectrophoretic Cell Classification Method". The method of this patent takes advantage of the principle of dielectrophoresis which is employed in, for example, the petroleum industry where it is used to extract sulphur from crude oil.
As applied to biological systems, the process disclosed in the patent to Pohl subjected cells to a non-uniform electric field between two electrodes. Dielectrophoresis is said to cause cells to separate from one another and then be collected in two separate chambers. The shortcomings of this method were that Pohl had no means to make a final physical separation of the stream without turbulence and subsequent remixing of the elements before they entered the final reservoirs. In actuality, he never achieved this separation. The patent does not address the necessity of carrying the cells in a deionized solution, nor the necessity of using a high frequency alternating field. Finally, the shape of the Pohl tube does not allow for the spreading of the cells in the field because of its uniform configuration throughout that eliminates the adjustability and fine tuning required for quality sorting.
Accordingly, a method of sorting cells with a high degree of differentiation and viability is still wanting. This need has not been met by earlier efforts to overcome the common problems in sorting cells.